RNA Interference Targeting To Platelet-derived Growth Factor Receptor β Subunit In Hepatic Stellate Cells Ameliorates Experimental Hepatic Fibrosis In Rats

Background and ObjectiveHepatic fibrosis,characterized by the overproduction and accumulation of hepatic extracellular matrix(ECM),was the common and important pathological changes in the chronic liver diseases,through which the chronic hepatitis developed into cirrhosis.It was acknowledged that hepatic cirrhosis couldn't be reversed,while hepatic fibrosis could. Hepatic stellate cell(HSC) was the main resource of hepatic ECM and the cellular basis for hepatic fibrosis.The activation of HSC was recognized as the key event of the pathogenesis of hepatic fibrosis.Inhibiting the activation,proliferation and migration of HSCs is one of the most important strategies to prevent the hepatic fibrogenesis.In fibrotic liver,PDGF is the most potent mitogen for HSCs.PDGF binds to structurally related tyrosine kinase receptors that is a homodimer or heterodimer ofαorβsubunits.PDGFR-αis constitutively expressed in quiescent HSCs,whereas PDGFR-βis acquired as the activation of HSCs,the major cell type expressing PDGFR-βin liver. PDGF and PDGFR-βplay important roles in hepatic fibrogenesis,therefore,targeting to PDGF,PDGF receptor and molecules related to PDGF intracellular signaling transduction pathway is attractive for therapeutic intervention in hepatic fibrosis.RNAi(RNA interference) is one of the gene-silencing technologies,which is considered as an antisense mechanism of action that utilizes double strand RNase to promote hydrolysis of target RNA.In this study,we inhibited the PDGFR-βexpression by small interference RNA(siRNA) to block the binding of PDGF,and investigated its effect on biological characteristics of HSCs in vitro.Furthermore,we constructed a PDGFR-βshRNA(short hairpin RNA) expression vector and investigated its antifibrogenic effect on experimental hepatic fibrosis.To avoid the non-specific interference of PDGFR-βgene expression in other tissues and cells,we used cell-specific glial fibrillary acidic protein (GFAP) promoter to drive the expression of PDGFR-βshRNA in HSCs of liver,and investigated its antifibrogenic effect in vivo.Methods1.Screening for the PDGFR-βsiRNA with high gene-silencing efficacy The targeted region of siRNA was the coding sequence of PDGFR-βcDNA.We selected 3 pairs 21-nucleotide RNAs and 1 pairs non-specific nucleotide RNA.Transient transfection of siRNA by lipofection method was carried out in the rat HSC-T6 cell line, whose phenotype was activated HSC.Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expressions of PDGFR-β.2.Investigate the effect of PDGFR-βsiRNA on biological characteristics of HSCCells were transfected with PDGFR-βsiRNA and were stimulated by PDGF-BB,and then were collected.RT-PCR was used to detect the expression of fibrogenesis- related genes.BrdU incorporation was performed to determine cell proliferation.Annexin V/PI staining method was utilized to measure cell apoptosis.Mitogen-activated protein kinases (MAPK) signaling transduction pathway was necessary for the HSC proliferation induced by PDGF,and extracellular-signal- related kinase(ERK) phosphorylation and c-fos expression were the two important processes of MAPK signaling transduction pathway, which were detected by Western Blot and RT-PCR in this study.3.Construct plasmid expressing PDGFR-βshRNAThe disadvantages of chemically synthesized siRNA,such as instability and easy to be degraded by serum nucleases restrict its application in vivo.To investigate the effect of PDGFR-βsiRNA on experimental hepatic fibrosis,we inserted PDGFR-βshRNA into the miR30-based shRNA expression system of pCMR30 Vector to generate pCMV-shRNA-LacZ,in which PDGFR-βshRNA was driven by CMV promoter andβ-galactosidase(β-gal) was the reporter gene.As a negative control,we generated pCMV-NC-LacZ with miR30-based PDGFR-βshRNA in pCMV-shRNA- LacZ replaced by negative control RNA.4.Investigate the effect of pCMV-shRNA-LacZ on experimental hepatic fibrosisRats model of hepatic fibrosis were induced by common bile duct ligation(BDL) and dimethylnitrosamine(DMN) treatment,pCMV-shRNA-LacZ solutions were injected once every 5 days with hydrodynamics-based transfection method.to induce stable expression of PDGFR-βshRNA.After the rats models were constructed successfully,rats were sacrificed,serum samples were collected for biochemical tests and liver tissues were taken out for histology and Western Blot analysis.5.Construct plasmid expressing PDGFR-βshRNA driven by GFAP promoterThe GFAP promoter in the plasmid named pGfa2-clac was PCR-cloned into the EcoRI and BamHI site and replaced the CMV promoter of pCMV-shRNA-LacZ to generate pGfa-shRNA-LacZ,pGfa-NC-LacZ was generated by replacement of PDGFR-βshRNA in pGfa-shRNA-LacZ with non-specific nucleotide RNA as a negative control.All the constructed plasmids were verified by PCR and sequencing.6.Locate LacZ gene expression in acute and chronic liver injury rats modelTo locate LacZ reporter gene expression driven by GFAP promoter in liver,we injected pGfa-shRNA-LacZ or pCMV-shRNA-LacZ by hydrodynamics-based transfection method into normal rats,acute liver injury rats induced by CCl4 and chronic liver injury rats induced by BDL.Twenty four hours after plasmids injection,all rats were anesthetized and perfused,livers were taken out and postfixed.Immunofluorescence and laser scanning confocal techniques were used to observe the distribution ofβ-gal protein,and the colocalization ofβ-gal andα-SMA orβ-gal and GFAP.7.Investigate the effect of pGfa-shRNA-LacZ on experimental hepatic fibrosisHepatic fibrosis was induced by BDL in rats,pGfa-shRNA-LacZ solutions were injected once every 5 days with hydrodynamics-based transfection method.to induce stable expression of PDGFR-βshRNA.After the rats models were constructed successfully,rats were sacrificed,serum samples were collected for biochemical tests and liver tissues were taken out for histology and hydroxyproline content test.Results1.PDGFR-βsiRNA could downregulate PDGFR-βexpressionAll the 3 pairs of siRNAs could suppress PDGFR-βmRNA expression,especially siRNA₉₅₇,which had the highest gene-silencing efficacy.RT-PCR results showed that siRNA₉₅₇ downregulated the PDGFR-βmRNA expression by about(40±3.14)%, (55±4.67)%,(72±4.20)%and(55±6.19)%at 24h,48h,72h and 96h,respectively.In addition,PDGFR-βprotein expression was downregulated by about(33±4.51)%, (47±5.72)%,(60±7.59)%and(82±7.13)%at 24h,48h,72h and 96h,respectively.2.PDGFR-βsiRNA could suppress the activation and proliferation of HSCThe results of RT-PCR and Western Blot revealed that theα-SMA expression in HSCs was elevated significantly after PDGF-BB stimulation,which was impeded by PDGFR-βsiRNA.Furthermore,the upregulation of PDGF-B,CTGF,TIMP-1 and collagen typeâ… mRNA induced by PDGF-BB was eliminated by siRNA₉₅₇.However,the expressions of TGF-β₁ and MMP-2 mRNA in HSCs were not affected by PDGFR-βsiRNA₉₅₇ and/or PDGF-BB. 3.PDGFR-βsiRNA inhibited HSC proliferation induced by PDGF-BB,but did not affect the apoptosis of HSCTo investigate the effect of siRNA₉₅₇ on HSC proliferation,BrdU incorporation was used and analyzed by immunofluorescence and flow cytometry.Results showed that PDGF-BB significantly promoted the proliferation of HSC,which was inhibited by PDGFR-βsiRNA₉₅₇.Additionally,Western blot analysis revealed that the phosphorylation of ERK increased significantly after PDGF-BB stimulation,which was impeded by siRNA₉₅₇,RT-PCR analysis demonstrated that siRNA₉₅₇ suppressed the upregulation of transcription factor c-fos expression induced by PDGF-BB.Annexin V/PI flow cytometry analysis showed that neither PDGF-BB nor siRNA₉₅₇ could affect the percentage of apoptotic HSC.4.pCMV-shRNA-LacZ relieved liver injury and ameliorated hepatic fibrosis in ratsIn DMN animal model,results of serum biochemical tests revealed that serum total bilirubin(TB),alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels were elevated significantly in DMN control rats compared with the normal rats, which were reduced by pCMV-shRNA-LacZ treatment.In BDL animal model,serum TB, direct bilirubin(DB),ALT and AST levels were elevated significantly in BDL control group compared with the sham-operated group,which was eliminated by pCMV-shRNA-LacZ treatment.HE staining and Masson's trichrome staining of liver sections showed that liver injury and hepatic fibrosis in both rats models were ameliorated by pCMV-shRNA-LacZ treatment.Western Blot analysis indicated that both PDGFR-βandα-SMA protein levels were elevated significantly in DMN treatment rats and BDL rats, and remarkably reduced by pCMV-shRNA-LacZ treatment.The results were confirmed by immunohistochemical examination.5.GFAP promoter targets LacZ express into HSCImmunofluorescence analysis indicated thatβ-gal-positive cells were all distributed in the pericentral and portal area of hepatic lobule in normal rats,acute liver injury rats and chronic liver injury rats for both pCMV-shRNA-LacZ and pGfa-shRNA-LacZ treatment. The results also revealed thatβ-gal proteins were detected in both hepatocytes and HSC, which were positive forα-SMA and/or GFAP staining in the liver treated with pCMV-shRNA-LacZ.While in the liver treated with pGfa-shRNA-LacZ,β-gal proteins were co-expressed with GFAP and/orα-SMA,and were located in the perisinusoidal space, suggesting that GFAP promoter targeted the reporter gene LacZ specifically expressing in HSC and CMV promoter directed LacZ expressing in not only HSCs,but also hepatocytes.6.pGfa-shRNA-LacZ relieved liver injury and ameliorated hepatic fibrosis in ratsBiochemical tests indicated that the elevated serum levels of TB,indirect bilirubin (IDB),ALT and AST induced by BDL could be markedly reduced by pGfa- shRNA-LacZ treatment.Immunohistochemical and Masson's trichrome staining of liver sections showed significant collagen deposition and bile duct hyperplasia in BDL rats,and increased protein expressions of PDGFR-βandα-SMA were also observed in them,while both the hepatic fibrosis and the increased expressions of PDGFR-βandα-SMA protein were significantly relieved by pGfa-shRNA-LacZ treatment.In addition,the hydroxyproline content increased more than 2-fold in BDL rats compared with the sham-operated rats,while pGfa-shRNA-LacZ treatment decreased about half of the hydroxyproline content in BDL rats.Conclusion1.siRNA against PDGFR-βcould block the binding of PDGF,inhibit the MAPK signaling transduction pathway and suppress HSC proliferation consequently.Furthermore, the activation and ECM production of HSC stimulated by PDGF-BB could also be inhibited by PDGFR-βsiRNA.2.PDGFR-βsiRNA is active as an antifibrogenic gene therapeutic method able to reduce the expression of PDGFR-βprotein and ameliorated liver injury and hepatic fibrosis induced by BDL and DMN.3.GFAP promoter was able to target genes downstream express into HSC.4.PDGFR-βshRNA driven by GFAP promoter could be targeted into HSC,and could inhibit the activation of HSC,relieve liver injury and hepatic fibrosis induced by BDL.